- Email: [email protected]
Altered circulating neurokinin B (NKB) levels are not due to sequence variants in the encoding genes
Letters to the Editor / European Journal of Obstetrics & Gynecology and Reproductive Biology 140 (2008) 124–148
low threshold for the diagnosis of PID.’’ [1] Therefore, gynecologists must recognize non-gynecologic conditions which fulfill minimal diagnostic criteria and mimic PID. Acute epiploic appendagitis is a self-limiting inflammation of the appendices epiploicae, known to mimic appendicitis and acute diverticulitis. CT scan findings of a 1.5–3.5 cm oval shaped, fat density lesion that abuts the anterior sigmoid colon wall, surrounded by inflammatory change, are thought to be pathognomonic for epiploic appendagitis [3]. Before the widespread advent of CT imaging, it was usually diagnosed at surgery. The etiology is thought to be torsion of epiploic appendages, with resultant vascular occlusion leading to ischemia. Treatment is conservative with oral anti-inflammatory medications, and antibiotics are not indicated. Surgery is not indicated and most patients recover with conservative management in less than 10 days. Misdiagnosis of acute epiploic appendagitis as PID may result in unnecessary hospital admissions and excessive use of broad-spectrum antibiotic therapy. This is the first report describing epiploic appendagitis as a mimic of PID.
References [1] Centers for Disease Control and Prevention, Workowski KA, Berman SM. Sexually transmitted diseases treatment guidelines, 2006. MMWR Recomm Rep 2006;55(RR-11):1–94. [2] Sandrasegaran K, Maglinte DD, Rajesh A, et al. Primary epiploic appendagitis: CT diagnosis. Emerg Radiol 2004;11(1):9–14. [3] Singh AK, Gervais DA, Hahn PF, et al. Acute epiploic appendagitis and its mimics. Radiographics 2005;25(6):1521–34.
Alexander M. Quaas* Department of Obstetrics and Gynecology, Massachusetts General Hospital, Boston, MA, United States Peter R. Mueller Department of Radiology, Massachusetts General Hospital, Boston, MA, United States Jennifer M. Kickham Department of Obstetrics and Gynecology, Massachusetts General Hospital, Boston, MA, United States *Corresponding author. Tel.: +1 617 7249030; fax: +1 617 7264267 E-mail address: [email protected] (A.M. Quaas) 21 April 2007 doi:10.1016/j.ejogrb.2007.09.002
135
Altered circulating neurokinin B (NKB) levels are not due to sequence variants in the encoding genes Dear Editor, The measurement of elevated levels of neurokinin B (NKB) in pregnancies complicated by pre-eclampsia has been confirmed in several studies [1]. To investigate whether DNA sequence variants could possibly underlie the alteration in circulating NKB levels or predispose to pre-eclampsia, we performed mutation screening of the TAC3 and TACR3 genes, which encode neurokinin B and its receptor (NK3), respectively, in 140 patients with early-onset severe pre-eclampsia [2] and 60 control individuals. Concurrently, NKB levels were measured by EIA in 52 of the patient samples. Genomic amplicons representing all the exons (with flanking intronic regions, some extending into the 50 and 30 untranslated regions) were subjected to Multiphor single stranded conformational polymorphism (SSCP)/heteroduplex analysis (HA). Conformational variants were further characterised by bidirectional automated sequencing and subsequently, genotyping was performed by restriction enzyme analysis, where appropriate. Five sequence variants were identified (Table 1) including three which had previously been documented. A novel intronic TAC3 base pair substitution (G-T) was identified 53 bp before the start of exon 4. There was no statistically significant association demonstrated in the comparison of genotype frequencies in patient and control groups ( p = 0.47). Despite the variant being localised beyond the consensus intron-exon splice boundary, and not within a recognized splice branch site, it could still create a splicing defect. This could result in an alteration in the level of the mature mRNA generated, and consequently contribute to disease, reported to be the case for 15% of all intronic polymorphisms [3]. The second novel variant was a T-C base pair substitution in the third nucleotide of TAC3 exon 5 (representing coding nucleotide position 295 of the gene). The variant was identified at similar frequencies in patients and controls (0.14 and 0.10, respectively) and no significant association was demonstrated in the comparison of the two groups ( p = 0.93). The effect of this sequence variant is a change from a hydrophilic serine to a hydrophobic proline amino acid residue. Application of the ESEfinder program (http://exon.cshl.edu/ESE) predicted that in the presence of the variant C allele, single SC35 and SRp40 binding sites are abolished, while a novel one is created. This provides in silica evidence for an increase in the potential of alternative splicing and needs to be addressed in functional studies. Of the previously documented sequence variants identified in this study, the TACR3 857A-G [dbSNP: rs2276973] is rare and TAC3-25 C-T [dbSNP:rs2291855] and TACR3 -103 T-C [dbSNP: rs3733632] are 50 untranslated region polymorphisms. A statistical significant difference, p = 0.02,
136
Letter to the EditorLetters to the Editor / European Journal of Obstetrics & Gynecology and Reproductive Biology 140 (2008) 124–148
Table 1 Genotype and allele frequencies of the loci identified in the TAC3 and TACR3 genes in pre-eclampsia and control individuals Gene and position TAC3 g.-25 C ! T promoter
Genotype
CC CT TT C T
IVS3-53 G ! Ta intronic
GG GT TT G T
g.295 T ! C a exonic
TT TC CC T C
TACR3 g.-103T ! C promoter
g.857 A ! G exonic
a
PE patients n = 140
Controls n = 60
117 (0.83) 20 (0.15) 3 (0.02)
51 (0.85) 9 (0.15) 0
0.90 0.10 135 (0.96) 5 (0.04) 0 0.98 0.02 120 (0.86) 20 (0.14) 0 0.93 0.07
p-Value
References
0.39
0.92 0.08 59 (.099) 1 (0.01) 0
0.47
1.0 0 54 (0.90) 6 (0.10) 0
genetic variants in the TAC3 and TACR3 genes as a major cause of altered circulating NKB level.
0.93
0.95 0.05
TT TC CC T C
6 (0.05) 50 (0.42) 64 (0.53) 0.26 0.74
9 (0.15) 29 (0.48) 22 (0.37) 0.39 0.61
0.02
AA AG GG A G
111 (0.93) 9 (0.07) 0 0.96 0.04
57 (0.95) 3 (0.05) 0 0.97 0.03
0.52
[1] Zulfikaroglu E, Ugur M, Taflan S, et al. B levels in maternal and umbilical cord blood in preeclamptic and normal pregnancies. J Perinat Med 2007;35(3):200–2. [2] Davey DA, MacGillivray I. The classification and definition of the hypertensive disorders of pregnancy. Am J Obstet Gynecol 1988;158(4):892–8. [3] Cartegni L, Chew SL, Krainer AR. Listening to silence and nonsense mutations: exonic mutations that affect splicing. Nat Rev 2002;3(22):198–285. [4] Hoogendoom B, Coleman SL, Guy CA, et al. Functional analysis of human promoter polymorphisms. Hum Mol Genet 2003;12(18): 2249–54. [5] Kunika K, Tanahashi T, Kudo E, et al. Effect of +36T>C in intron 1 on the glutamine:fructose-6-phosphate amoditransferase 1 gene and its contribution to type 2 diabetes in different populations. J Hum Genet 2006;51:1100–9. [6] Geissbuehler V, Moser R, Zimmermann K, et al. Arch Gynecol Obstet 2007. doi:10.1007/s00404-006-0316-y.
Kashefa Carelse Tofa Stefan Gebhardt Department of Obstetrics and Gynaecology, University of Stellenbosch, South Africa George Rebello Division of Human Genetics, University of Cape Town, South Africa
Novel variant.
was observed in the comparison of patient and control genotypes at the TACR3-103 T-C locus. Variation within this region is likely to influence gene transcription, which in turn could lead to altered gene expression. Examples of this phenomenon include uroporphyrinogen III synthase erythroid promoter variants, which cause erythropoeitic porphyria and a single IL-3 promoter region SNP associated with rheumatoid arthritis. One third of promoter variants exert a functional effect on gene expression [4]. It is recommended that the effect of the TACR3-103 T-C promoter variant be further investigated by using a Luciferase reporter transfection study [5]. A very broad spectrum of NKB level was measured in the 52 patient samples (9.6–267.8 ng/L) [6]. No association could be demonstrated between NKB level range and any particular genotype or haplotype in the patient or the control groups. The lack of clear relationship between any of these polymorphisms and circulating NKB level suggests that regulation of these genes’ altered expression in preeclampsia may occur at a level other than that of transcription. Collectively, these data confirm the genetic heterogeneity of pre-eclampsia and discount the role of
Verena Geissbuehler Frauenklinik Kantonsspital, Frauenfeld, Switzerland Rene Moser IBR-Inc. Laboratories, Matzingen, Switzerland Megan Stolk Renate Hillermann* Department of Genetics, University of Stellenbosch, South Africa *Corresponding author. Tel.: +27 21 808 5824; fax: +27 21 808 5833 E-mail address: [email protected] (R. Hillermann) 24 April 2007 doi:10.1016/j.ejogrb.2007.09.003
low threshold for the diagnosis of PID.’’ [1] Therefore, gynecologists must recognize non-gynecologic conditions which fulfill minimal diagnostic criteria and mimic PID. Acute epiploic appendagitis is a self-limiting inflammation of the appendices epiploicae, known to mimic appendicitis and acute diverticulitis. CT scan findings of a 1.5–3.5 cm oval shaped, fat density lesion that abuts the anterior sigmoid colon wall, surrounded by inflammatory change, are thought to be pathognomonic for epiploic appendagitis [3]. Before the widespread advent of CT imaging, it was usually diagnosed at surgery. The etiology is thought to be torsion of epiploic appendages, with resultant vascular occlusion leading to ischemia. Treatment is conservative with oral anti-inflammatory medications, and antibiotics are not indicated. Surgery is not indicated and most patients recover with conservative management in less than 10 days. Misdiagnosis of acute epiploic appendagitis as PID may result in unnecessary hospital admissions and excessive use of broad-spectrum antibiotic therapy. This is the first report describing epiploic appendagitis as a mimic of PID.
References [1] Centers for Disease Control and Prevention, Workowski KA, Berman SM. Sexually transmitted diseases treatment guidelines, 2006. MMWR Recomm Rep 2006;55(RR-11):1–94. [2] Sandrasegaran K, Maglinte DD, Rajesh A, et al. Primary epiploic appendagitis: CT diagnosis. Emerg Radiol 2004;11(1):9–14. [3] Singh AK, Gervais DA, Hahn PF, et al. Acute epiploic appendagitis and its mimics. Radiographics 2005;25(6):1521–34.
Alexander M. Quaas* Department of Obstetrics and Gynecology, Massachusetts General Hospital, Boston, MA, United States Peter R. Mueller Department of Radiology, Massachusetts General Hospital, Boston, MA, United States Jennifer M. Kickham Department of Obstetrics and Gynecology, Massachusetts General Hospital, Boston, MA, United States *Corresponding author. Tel.: +1 617 7249030; fax: +1 617 7264267 E-mail address: [email protected] (A.M. Quaas) 21 April 2007 doi:10.1016/j.ejogrb.2007.09.002
135
Altered circulating neurokinin B (NKB) levels are not due to sequence variants in the encoding genes Dear Editor, The measurement of elevated levels of neurokinin B (NKB) in pregnancies complicated by pre-eclampsia has been confirmed in several studies [1]. To investigate whether DNA sequence variants could possibly underlie the alteration in circulating NKB levels or predispose to pre-eclampsia, we performed mutation screening of the TAC3 and TACR3 genes, which encode neurokinin B and its receptor (NK3), respectively, in 140 patients with early-onset severe pre-eclampsia [2] and 60 control individuals. Concurrently, NKB levels were measured by EIA in 52 of the patient samples. Genomic amplicons representing all the exons (with flanking intronic regions, some extending into the 50 and 30 untranslated regions) were subjected to Multiphor single stranded conformational polymorphism (SSCP)/heteroduplex analysis (HA). Conformational variants were further characterised by bidirectional automated sequencing and subsequently, genotyping was performed by restriction enzyme analysis, where appropriate. Five sequence variants were identified (Table 1) including three which had previously been documented. A novel intronic TAC3 base pair substitution (G-T) was identified 53 bp before the start of exon 4. There was no statistically significant association demonstrated in the comparison of genotype frequencies in patient and control groups ( p = 0.47). Despite the variant being localised beyond the consensus intron-exon splice boundary, and not within a recognized splice branch site, it could still create a splicing defect. This could result in an alteration in the level of the mature mRNA generated, and consequently contribute to disease, reported to be the case for 15% of all intronic polymorphisms [3]. The second novel variant was a T-C base pair substitution in the third nucleotide of TAC3 exon 5 (representing coding nucleotide position 295 of the gene). The variant was identified at similar frequencies in patients and controls (0.14 and 0.10, respectively) and no significant association was demonstrated in the comparison of the two groups ( p = 0.93). The effect of this sequence variant is a change from a hydrophilic serine to a hydrophobic proline amino acid residue. Application of the ESEfinder program (http://exon.cshl.edu/ESE) predicted that in the presence of the variant C allele, single SC35 and SRp40 binding sites are abolished, while a novel one is created. This provides in silica evidence for an increase in the potential of alternative splicing and needs to be addressed in functional studies. Of the previously documented sequence variants identified in this study, the TACR3 857A-G [dbSNP: rs2276973] is rare and TAC3-25 C-T [dbSNP:rs2291855] and TACR3 -103 T-C [dbSNP: rs3733632] are 50 untranslated region polymorphisms. A statistical significant difference, p = 0.02,
136
Letter to the EditorLetters to the Editor / European Journal of Obstetrics & Gynecology and Reproductive Biology 140 (2008) 124–148
Table 1 Genotype and allele frequencies of the loci identified in the TAC3 and TACR3 genes in pre-eclampsia and control individuals Gene and position TAC3 g.-25 C ! T promoter
Genotype
CC CT TT C T
IVS3-53 G ! Ta intronic
GG GT TT G T
g.295 T ! C a exonic
TT TC CC T C
TACR3 g.-103T ! C promoter
g.857 A ! G exonic
a
PE patients n = 140
Controls n = 60
117 (0.83) 20 (0.15) 3 (0.02)
51 (0.85) 9 (0.15) 0
0.90 0.10 135 (0.96) 5 (0.04) 0 0.98 0.02 120 (0.86) 20 (0.14) 0 0.93 0.07
p-Value
References
0.39
0.92 0.08 59 (.099) 1 (0.01) 0
0.47
1.0 0 54 (0.90) 6 (0.10) 0
genetic variants in the TAC3 and TACR3 genes as a major cause of altered circulating NKB level.
0.93
0.95 0.05
TT TC CC T C
6 (0.05) 50 (0.42) 64 (0.53) 0.26 0.74
9 (0.15) 29 (0.48) 22 (0.37) 0.39 0.61
0.02
AA AG GG A G
111 (0.93) 9 (0.07) 0 0.96 0.04
57 (0.95) 3 (0.05) 0 0.97 0.03
0.52
[1] Zulfikaroglu E, Ugur M, Taflan S, et al. B levels in maternal and umbilical cord blood in preeclamptic and normal pregnancies. J Perinat Med 2007;35(3):200–2. [2] Davey DA, MacGillivray I. The classification and definition of the hypertensive disorders of pregnancy. Am J Obstet Gynecol 1988;158(4):892–8. [3] Cartegni L, Chew SL, Krainer AR. Listening to silence and nonsense mutations: exonic mutations that affect splicing. Nat Rev 2002;3(22):198–285. [4] Hoogendoom B, Coleman SL, Guy CA, et al. Functional analysis of human promoter polymorphisms. Hum Mol Genet 2003;12(18): 2249–54. [5] Kunika K, Tanahashi T, Kudo E, et al. Effect of +36T>C in intron 1 on the glutamine:fructose-6-phosphate amoditransferase 1 gene and its contribution to type 2 diabetes in different populations. J Hum Genet 2006;51:1100–9. [6] Geissbuehler V, Moser R, Zimmermann K, et al. Arch Gynecol Obstet 2007. doi:10.1007/s00404-006-0316-y.
Kashefa Carelse Tofa Stefan Gebhardt Department of Obstetrics and Gynaecology, University of Stellenbosch, South Africa George Rebello Division of Human Genetics, University of Cape Town, South Africa
Novel variant.
was observed in the comparison of patient and control genotypes at the TACR3-103 T-C locus. Variation within this region is likely to influence gene transcription, which in turn could lead to altered gene expression. Examples of this phenomenon include uroporphyrinogen III synthase erythroid promoter variants, which cause erythropoeitic porphyria and a single IL-3 promoter region SNP associated with rheumatoid arthritis. One third of promoter variants exert a functional effect on gene expression [4]. It is recommended that the effect of the TACR3-103 T-C promoter variant be further investigated by using a Luciferase reporter transfection study [5]. A very broad spectrum of NKB level was measured in the 52 patient samples (9.6–267.8 ng/L) [6]. No association could be demonstrated between NKB level range and any particular genotype or haplotype in the patient or the control groups. The lack of clear relationship between any of these polymorphisms and circulating NKB level suggests that regulation of these genes’ altered expression in preeclampsia may occur at a level other than that of transcription. Collectively, these data confirm the genetic heterogeneity of pre-eclampsia and discount the role of
Verena Geissbuehler Frauenklinik Kantonsspital, Frauenfeld, Switzerland Rene Moser IBR-Inc. Laboratories, Matzingen, Switzerland Megan Stolk Renate Hillermann* Department of Genetics, University of Stellenbosch, South Africa *Corresponding author. Tel.: +27 21 808 5824; fax: +27 21 808 5833 E-mail address: [email protected] (R. Hillermann) 24 April 2007 doi:10.1016/j.ejogrb.2007.09.003